Enzyme-linked Immunosorbent assay of Escherichia coli O157 : H7 in surface enhanced Poly(Methyl methacrylate) microchannels

摘要

A novel surface treatment method was developed to enhance polymer-based microchannel enzyme-linked immunosorbent assay (ELISA) for Escherichia coli 0157:H7 detection. By applying an amine-bearing polymer, poly(ethyleneimine) (PEI), onto poly(methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli 0157:1-17 detection. Compared with untreated PMMA microchannels, similar to 45 times higher signal and 3 times higher signaloise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to similar to 2 min, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5-8 cells per sample, which is also better than 15-30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli 0157:H7.